Introduction to Confocal Laser Scanning Microscopy (LEICA)

[Pages:87]Introduction to Confocal Laser Scanning

Microscopy (LEICA)

This presentation has been put together as a common effort of Urs Ziegler, Anne Greet Bittermann, Mathias Hoechli. Many pages are copied from Internet web pages or from presentations given by Leica, Zeiss and other companies. Please browse the internet to learn interactively all about optics. For questions & registration please contact zmb.unizh.ch .

Confocal Laser Scanning Microscopy

xy

yz

xz

110000 ??mm

xy

yz xz

thick specimens at different depth

3D reconstruction

Types of confocal microscopes

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point confocal

slit confocal

spinning disc confocal (Nipkov)

Best resolution and out-of-focus suppression as well as highest multispectral flexibility is achieved only by the classical single point confocal system !

Fundamental Set-up of Fluorescence Microscopes:

confocal vs. widefield

Confocal Fluorescence Microscopy

Widefield Fluorescence

Microscopy

LASER

Light source pinhole aperture

Photomultiplier detector

Detector pinhole aperture

Dichroic mirror

Okular

Objectives Sample Plane

Fluorescence Filter Cube

Z Focus

CCD

Fluorescence Light Source

Confocal laser scanning microscope - set up:

The system is composed of a a regular florescence microscope and the confocal part, including scan head, laser optics, computer.

Comparison: Widefield - Confocal

Y X

Higher z-resolution and reduced out-of-focus-blur make confocal pictures crisper and clearer. Only a small volume can be visualized by confocal microscopes at once. Bigger volumes need time consuming sampling and image reassembling.

Comparison: Widefield - Confocal

optical resolution in z

Widefield Confocal

2 - 3 ?m 0.5 ?m

Comparison: Widefield - Confocal

region of out-of-focusinformation

Widefield Confocal

blurred & large

very small

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